Mass spectrometry study of N-alkylbenzenesulfonamides with potential antagonist activity to potassium channels

Article abstract of DOI:10.1007/s00726-015-2099-6

Herein, we report the synthesis and mass spectrometry studies of several N-alkylbenzenesulfonamides structurally related to sulfanilic acid. The compounds were synthesized using a modified Schotten-Baumann reaction coupled with Meisenheimer arylation. Sequential mass spectrometry by negative mode electrospray ionization (ESI(-)-MS/MS) showed the formation of sulfoxylate anion (m/z 65) observed in the mass spectrum of p-chloro-N-alkylbenzenesulfonamides. Investigation of the unexpected loss of two water molecules, as observed by electron ionization mass spectrometry (EI-MS) analysis of p-(N-alkyl)lactam sulfonamides, led to the proposal of corresponding fragmentation pathways. These compounds showed loss of neutral iminosulfane dioxide molecule (M-79) with formation of ions observed at m/z 344 and 377. These ions were formed by rearrangement on ESI(+)-MS/MS analysis. Some of the molecules showed antagonistic activity against Kv3.1 voltage-gated potassium channels.

Full text of DOI:10.1007/s00726-015-2099-6

Amino Acids
DOI 10.1007/s00726-015-2099-6
ORIGINAL ARTICLE
Mass spectrometry study of N‑alkylbenzenesulfonamides
with potential antagonist activity to potassium channels
Carina C. Martins1 · Carlos A. Zanutto Bassetto Jr.1 · Jandyson M. Santos2 ·
Marcos N. Eberlin2 · Alvicler Magalhães3 · Wamberto Varanda4 ·
Eduardo R. Perez Gonzalez1
Received: 7 September 2015 / Accepted: 9 September 2015
© Springer-Verlag Wien 2015
Abstract Herein, we report the synthesis and mass spec-
trometry studies of several N-alkylbenzenesulfonamides
structurally related to sulfanilic acid. The compounds
were synthesized using a modified Schotten–Baumann
reaction coupled with Meisenheimer arylation. Sequen-
tial mass spectrometry by negative mode electrospray
ionization (ESI(−)-MS/MS) showed the formation of sul-
foxylate anion (m/z 65) observed in the mass spectrum of
p-chloro-N-alkylbenzenesulfonamides. Investigation of
the unexpected loss of two water molecules, as observed
by electron ionization mass spectrometry (EI-MS) analy-
sis of p-(N-alkyl)lactam sulfonamides, led to the pro-
posal of corresponding fragmentation pathways. These
compounds showed loss of neutral iminosulfane dioxide
molecule (M-79) with formation of ions observed at m/z
344 and 377. These ions were formed by rearrangement on
ESI(+)-MS/MS analysis. Some of the molecules showed
antagonistic activity against Kv3.1 voltage-gated potas-
sium channels.
Keywords N-alkylbenzenesulfonamides · Mass
spectrometry · ESI-MS/MS · Kv3.1
Introduction
The benzene sulfonamides are amides of sulfanilic acid,
characterized by the presence of Ar–SO2–NR′R″ groups.
The most widely used synthetic method for the prepara-
tion of sulfonamides is by using Schotten–Baumann reac-
tion conditions, which involves the condensation of acid
chloride with an amine in the presence of a base (Deng and
Mani 2006; Smith and March 2001). These compounds are
known to be antibacterial agents (Joshi and Khosla 2003)
and are also reported to inhibit the activity of carbonic
anhydrase and cyclooxygenases (COX-1 and COX-2)
(Supuran et al. 1998; Dannhardt and Kiefer 2001; Li et al.
1995). They also have anticancer (Scozzafava et al. 2002),
anti-HIV (Bromidge et al. 2002), antifungal and antituber-
culosis (Goodman 1996) activity.
Handling Editor: J. Leban.
Electronic supplementary material The online version of this
article (doi:10.1007/s00726-015-2099-6) contains supplementary
material, which is available to authorized users.
* Eduardo R. Perez Gonzalez
eperez@fct.unesp.br
1
Laboratório de Química Orgânica Fina, Departamento
de Física, Química e Biologia, Faculdade de Ciências e
Tecnologia, Universidade Estadual Paulista (UNESP),
C.P. 467, Presidente Prudente, SP 19060-900, Brazil
The structure of sulfonamides is very similar to para-
aminobenzoic acid (PABA), and they inhibit the bacterial
growth and multiplication by competition with PABA for
the active site of the dihydropteroate synthase enzyme,
which is an essential precursor for bacterial synthesis
of folic acid, an important precursor in the synthesis of
nucleic acids (Supuran et al. 1998). PABA derivatives and
analogs are known to be local anesthetics (LA) (Gonçalves
et al. 2011) and block nerve conduction, reversibly binding
2
ThoMSon Mass Spectrometry Laboratory, Institute
of Chemistry, University of Campinas, UNICAMP,
Campinas, SP 13083-970, Brazil
3
Departamento de Química Inorgânica, Instituto de
Química, Universidade de Campinas, UNICAMP,
Campinas 13083-970, SP, Brazil
4
Department of Physiology, School of Medicine of Ribeirão
Preto, University of São Paulo, Ribeirão Preto, SP
14049-900, Brazil
1 3

C. C. Martins et al.
Scheme 1 Synthesis of
R
HN
NH2
O
p-chloro-N-alkylbenzenesulfon-
amides (1–8) and p-(N-alkyl)
lactam sulfonamides (9–20)
O S O
N
n
R
Cl
O S O
HN
NO2
HN
APP (n = 1)
APA (n = 3)
O S O
MeCN
r.t. 2h
DMSO
r.t. 12h, 85ºC
H2N R
+
X
X
N
O
Cl
n
Cl
X= NO2, H
1-8
9-20
R = furfuryl, butyl, benzyl, ciclohexyl, 4-clorobenzyl and 2,4-diclorobenzyl.
to voltage-dependent sodium channels present in excitable
cell membranes (Gouauax and Mackinnon 2005).
were
N-(3-aminopropyl)-2-azepanone
(APA)
and
N-(3-aminopropyl)-2-pyrrolidinone (APP), which have
been described as inhibitors of human tryptase, an impor-
tant mediator in asthma (Zhao et al. 2004), and HIV-1 pro-
tease, respectively (Ghosh et al. 2009).
Ion channels are a class of transmembrane proteins
responsible for promoting the selective passage of ions,
such as sodium (Na+), potassium (K+), chloride (Cl−),
and calcium (Ca2+) (Gonçalves et al. 2011; Lü and Frank
2008). The selective movement of ions across cell mem-
branes is related to many physiological processes, such
as the control of the neurons and muscle excitability, the
generation and propagation of action potentials, release
of neurotransmitters, and setting of cardiac rhythms and
muscle tones. They also play an important role in hormone
secretion, cell volume regulation, gene expression, immune
response, and cell division. Ion channel dysfunctions are
involved in several pathological conditions such as hyper-
tension, pain, insomnia, diabetes, and constipation (Lü and
Frank 2008). In this context, it has been shown that mal-
functioning of voltage-dependent potassium channels (Kv)
is implicated in cardiac arrhythmia, epilepsy, multiple scle-
rosis, diabetes, and arthritis and thus can be taken as impor-
tant therapeutic targets (Wulf et al. 2009) for the control of
such pathological conditions. In the face of these findings,
the development of novel compounds whose therapeu-
tic action is directed to these types of voltage-dependent
ionic channels seems crucial. Benzene sulfonamides were
reported as potent and selective blockers of Kv1.5 (Gross
et al. 2007; Lloyd et al. 2007; Olsson et al. 2014) and its
use has been suggested in the treatment of atrial fibrilla-
tion (Wulf et al. 2009), the most common type of cardiac
arrhythmia. Recently, Olsson et al. (2014) reported a series
of lactam sulfonamides as potent inhibitors of Kv1.5 (Ye
et al. 2009).
All sulfonamides were studied by mass spectrometry
technique using ion fragmentation in the gas phase provid-
ing useful information regarding the reaction mechanism,
in addition to classical analyses using electron ionization
as ion source (EI-MS) (Irikura and Todua 2014). There are
some studies using electrospray ionization mass spectrom-
etry (ESI-MS) of the cationic sulfonamide derivatives (Sun
et al. 2007; Hu et al. 2010), and only recently investigations
into negative ion conditions have been reported (Hibbs
et al. 2013; Hu et al. 2008).
The techniques used in these studies are: mass spec-
trometry with electron ionization (EI-MS), high-resolution
mass spectrometry (HRMS) in the positive (ESI(+)-MS)
and negative (ESI(−)-MS) modes and tandem experiments
(ESI-MS/MS).
Results and discussion
Synthesis of N‑alkylbenzenesulfonamides
Synthesis of the p-chloro-N-alkylbenzenesulfonamides
(1–8) was performed by reaction between a benzenesulfo-
nyl chloride with different amines, according to the litera-
ture (Lü and Frank 2008). Because of the pharmacological
importance of the lactam group (Zhao et al. 2004; Ghosh
et al. 2009) and due to a recent report of Olsson et al.
(2014) about lactam sulfonamides as potent inhibitors of
Kv1.5 (Olsson et al. 2014) channels, the second step of this
work was to use p-chloro-N-alkylbenzenesulfonamides as
intermediate for the synthesis of p-(N-alkyl)lactam sulfona-
mides (9–20) (Scheme 1). The formation of p-(N-alkyl)
lactam sulfonamides was performed through a nucleophilic
Herein, we show the synthesis and chemical charac-
terization by different spectroscopic and spectrometric
techniques of several benzene sulfonamides with poten-
tial activity against potassium ion channels. A number
of synthesized sulfonamides have the N-(aminoalkyl)
lactam moiety in their structures. Lactam groups
1 3

Mass spectrometry study of N-alkylbenzenesulfonamides with potential antagonist activity to…
Fig. 1 EI-MS spectrum of
.
+
O
compounds 1 and 2
NH
HN
S
%
O
O
O
+
+
95
100
CH2
S
O
O
+
NO2
O
75
50
25
0
Cl
1
1a
NO2
NO2
MM = 316 g.mol-1
Cl
Cl
81
+
316
53
.
[M]
39
110
100
126
125
32
156
143
191
220
225
175
204
284
250
269
299
m/z
25
50
75
150
175
200
250
275
300
325
NH
O
O
O
HN
HN
O
%
96
O
S O
S O
100
75
50
25
0
CH2
S
O
O
O
Cl
Cl
2
-1
2
MM = 271 g.mol
Cl
MM = 271,72 g.mol-1
Cl
81
111
75
[M]
53
50
68
159
175
128
125
206
200
143
150
191
236
254
250
271
m/z
75
100
175
225
275
aromatic substitution reaction (SNAr) by modifying the
methodology of SNAr reactions previously described by
Gonçalves et al. (2011). Compounds 2 and 4 were also
reacted with amino lactams, but due to the absence of nitro
group no reaction occurred.
seems not to change the fragmentation mechanism of
p-chloro-N-alkylbenzenesulfonamides (1–8).
The ESI-MS in the negative ion mode of the compounds 1
and 2 allowed the observation of compounds as intact depro-
tonated molecules with their corresponding isotopic patterns
of the ions with m/z 315 and m/z 317 for 1 and m/z 270 and
m/z 272 for 2 (corresponding to chlorine isotope pattern). The
deprotonated molecules were selected and fragmented by
sequential MS experiments (ESI(−)-MS/MS). Spectrum of
1 showed the 4-chloro-3-nitrobenzyl anion of m/z 156 as the
100 %. Fragment anions of m/z 220 (4-chloro-3-nitrobenzene-
sulfonyl anion) and m/z 65 corresponding to sulfoxylate anion
were also observed (Fig. 2). On the other hand, spectrum of 2
showed sulfoxylate anion (m/z 65) as the base anion. Fragment
anions of m/z 111 and 175 were attributed to 4-chlorobenzyl
and 4-chlorobenzenesulfonyl anions, respectively (Fig. 3).
The fragmentation path shown by anionic sulfonamides
involved the formation of sulfoxylate ion of m/z 65 as
described in Fig. 2, differently from the loss of neutral mol-
ecule of sulfur dioxide (SO2) (Irikura and Todua 2014; Sun
et al. 2007; Hibbs et al. 2013; Lloyd et al. 2007).
Mass spectrometry study of the
p‑chloro‑N‑alkylbenzenesulfonamides (1–8)
An MS study was performed for p-chloro-N-alkylsulfon-
amides (1–8). As an example of the results obtained, two
cases can be discussed, compounds 4-chloro-3-nitro-N-fur-
furylbenzenesulfonamide (1) and 4-chloro-N-furfurylben-
zenesulfonamide (2). These compounds were chosen for
comparison of their MS spectra to know the influence of
the nitro group in the fragmentation mechanisms.
MS study of compounds 1 and 2 was performed by
EI-MS, ESI(−)-MS and ESI(−)-MS/MS. Figure 1 shows
the EI-MS spectrum of compound 1. Molecular ion was
observed at m/z 316. The base ion at m/z 95 corresponds
to furfurylimine. Furfuryl ion of m/z 81 and other fragment
ions were also observed in the spectrum.
In view of the differences between the fragments observed
(formation of sulfoxylate anion) and that reported by Hibbs
et al. (2013), it is possible to propose a fragmentation mech-
anism to explain the formation of the anion HSO2− with m/z
65. The proposed mechanism (Scheme 2) showed the loss
The EI-MS spectrum of compound 2 showed the same
fragmentation pattern as described for compound 1 (spec-
trum is shown in supplementary material) and, therefore,
the group at position 3 in the benzene ring (H or NO2)
1 3

C. C. Martins et al.
Fig. 2 ESI(−)-MS/MS spectra
of compounds 1 and 2
O
O
N+
O–
HN
O S O
Intens.
Cl
5
S
x10
O
O
5
4
3
2
1
0
155.98541
O
O
H
S
315
NO2
O
N+
O–
Cl
1
64.97031
Cl
MM = 316 g.mol-1
-
[M -H ]
219.94722
314.98355
m/z
80
160
240
320
400
O
S
O
O
O
O
H
S
Intens.
HN
S O
5
O
x10
64.96946
Cl
1
Cl
270
174.96131
0.8
Cl
110.99965
2
MM = 271 g.mol-1
0.4
0
-
[M-H]
269.99758
m/z
400
80
160
240
320
O
.
+
HN
O S O
O
O
.
HN
O S O
+
+
CH2
N
NO2
O
%
N
O
NH
98
100
HN
O S O
O
N
9
+
75
MM = 422 g.mol-1
CH2
N
O
50
25
0
O
N
.
81
+
[M - 2H2O]
N
142
70
.
387
+
[M]
304
53
50
207 226
130
186
281
160
150
320
370
423
100
200
250
300
350
400
m/z
Fig. 3 EI-MS spectrum of compound 9
1 3

Mass spectrometry study of N-alkylbenzenesulfonamides with potential antagonist activity to…
Scheme 2 The proposed
Cl
mechanism for the formation of
H
sulfoxylate anion (SO2H− with
H
O
S
O
H
m/z 65) of compound 2
C
O
N
S
m/z 65
O
S
O
O
O
O
HN
ion base
or
H
Cl
Cl
m/z 270
m/z 175
O
S
O
Cl
m/z 111
Scheme 3 The proposed
Cl
NO2
mechanism for the formation of
the sulfoxylate anion (SO2H−
with m/z 65) for compound 1
H
H
O
S
O
H
C
O
N
m/z 65
O
S
O
S
O
O
O
HN
H
or
O2N
O2N
Cl
Cl
m/z 315
m/z 220
O
S
O
O2N
Cl
m/z 156
ion base
of neutral furfurylimine molecule generating the fragment
anion of m/z 175, which was attributed to 4-chlorobenze-
nesulfonyl anion. This anion can undergo an intramolecular
rearrangement process resulting in a neutral loss of chlo-
robenzyne and formation of the sulfoxylate anion with m/z
65. The same fragment lost a neutral molecule of SO2 and
gave rise to the 4-chlorobenzyl anion of m/z 111 following
the mechanism reported by Hibbs et al. (2013).
Our proposed mechanism (Schemes 2, 3) explains the
behavior of a series of p-chloro-N-alkylsulfonamides under
ionization at negative mode conditions. The formation of
sulfoxylate anion was observed for all p-chloro-N-alkylsul-
fonamides (1–8). Synthesis and complete characterization
can be found in the Supplementary Material.
Mass spectrometry study of the p‑(N‑alkyl)
In the case of compound 2 (Scheme 2), where there is
not a strong π-electron withdrawing group (as the NO2
group) at position 3 of the benzene ring, the formation of
anion fragment m/z 65 (base ion in the spectrum of 2) and
the neutral loss of chlorobenzyne molecule are the most
favored processes.
In the spectrum of compound 1, the presence of nitro
group stabilized 2-chloronitrophenyl anion of m/z 156
(main anion) and the neutral loss of SO2 as reported by
Hibbs et al. (2013) (Scheme 3).
lactam sulfonamides (9–20)
Compounds 9 and 10 were chosen to exemplify the MS
study of compounds 9–20. EI-MS spectra of 9 and 10
(Figs. 3 and 4, respectively) show their respective molec-
ular ions of m/z 423 and m/z 451. The formation of the
base peak of m/z 98 to compound 9 and m/z 126 for com-
pound 10 involves the breaking of bonds in the alkyl chain
of the N-substituent (Fiedorow et al. 2005). The ion frag-
ments formed by the transfer of two hydrogen atoms via
1 3

C. C. Martins et al.
Fig. 4 EI-MS spectrum of
compound 10
O
HN
O
O
S
O
.
+
CH2
+
+
N
NH
O
.
+
NO2
O
O
%
100
N
NH
HN
S
+
126
96
O
O
CH2
O
10
HN
S
MM = 450 g.mol-1
O
O
O
75
50
25
0
81
O
N
.
N
+
170
[M - 2H2O]
O
N
114
415
N
.
+
320
[M]
207
223
304
254
356
350
387
400
451
50
100
150
200
250
300
450 m/z
Scheme 4 Mechanism of for-
mation of fragment ion of m/z
86 and m/z 114 observed in the
EI-MS spectrum of compounds
9 and 10, respectively (Fie-
dorow et al. 2005)
.
+
O
O
O
HN
O S O
HN
O S O
HN
O S O
OH
OH
H+
+
H
N
N
NO2
NO2
NO2
n
n
NH
H
NH
NH
m/z 86 (n = 1)
m/z 114 (n = 3)
.
H
H2C
+
.
N
N
O
O H
+
ion/neutral complex
n
n
McLafferty rearrangement (m/z 86 for 9 and m/z 114 for
10) were also observed. This type of double rearrangement
proceeds via formation of ion/neutral complex, followed
by abstraction of the hydrogen atom from the amino group
(Zhao et al. 2004; Fiedorow et al. 2005; Yamaoka et al.
2004) as shown in Scheme 4.
Interestingly, it was observed that the fragment ions
M−36 attributed to the loss of two water molecules (m/z
387 for 9 and m/z 415 for 10) by EI-MS analysis. The
loss of a water molecule is the result of so-called “ortho
effect” characteristic of the fragmentation of N-alkyl-
ortho-nitroanilines (Danikiewicz 1998). This fragmenta-
tion occurs from the rearrangement of a hydrogen atom
from the amino group to oxygen atom of the nitro group,
resulting in an sp2 bond between nitrogen and carbon. After
delocalization of negative charge, the transfer of the other
hydrogen atom (from α-alkyl carbon) occurs to oxygen of
the nitro group, resulting in a loss of the first water mol-
ecule (Danikiewicz 1998) generating the fragment M−18
(Scheme 5). The second loss of water molecule occurs by
the rearrangement of a hydrogen atom of the β-lactam car-
bon to the lactam oxygen atom via a McLafferty rearrange-
ment. After delocalization of negative charge, the hydrogen
is transferred from α-lactam carbon to carbonyl lactam, fol-
lowed by an elimination of the second water molecule gen-
erating the fragment ions M−32 of m/z 387 for 9 and m/z
415 for 10.
The fragment ions at m/z 304 and 320 present in both
compounds (independent of the lactam ring) correspond to
consecutive loss of water molecule (Fiedorow et al. 2005)
followed by a McLafferty rearrangement with formation
of three or four heterocyclic fragment members. The loss
of water molecule occurs by ortho effect characteristic of
the fragmentation of N-alkyl-ortho-nitroanilines (Danikie-
wicz 1998) generating the M−18 fragment as shown in
Scheme 5, followed by neutral loss of methyl lactam (m/z
304) or lactam (m/z 320). Scheme 6 summarizes the pos-
sibilities of fragmentation of the M−18 species.
1 3

Mass spectrometry study of N-alkylbenzenesulfonamides with potential antagonist activity to…
Scheme 5 The proposed
.
.
mechanism for consecutive
loss of two water molecules for
compounds 9 and 10
+
+
O
O
O
O
HN
O S O
HN
S
HN
S
HN
O S O
-
O
O
H
O
O
- H2O
-
+
+
O
O
+
O
O
N
N
N
N
N
N
O
O
N
O
OH
H
N
N
H
OH
-
H
H
N
N
N
O
O
n
n
n
n
[M - 18]
n = 1, m/z 423
n = 3, m/z 451
.
.
.
+
+
+
O
O
HN
O
HN
O S O
HN
O S O
O S O
- H2O
-
O
O
N
N O
N
N
N
H
N
H
n
H
N
N
N
OH
O H
+
H
H
n
n
n = 1, m/z 387
n = 3, m/z 415
The ESI-MS analysis in the positive ion mode of com-
pounds 9 and 10 allowed their detection as intact proto-
nated molecules with their corresponding isotopic patterns
with m/z 423 for 9 and m/z 451 for 10. The sodium cations
[M+Na]+ were also observed with m/z 445 and m/z 473,
respectively.
The protonated molecules were selected and fragmented
by sequential MS experiments (ESI(+)-MS/MS) (Fig. 5).
The base peak observed for both 9 and 10 was the furfuryl
ion of m/z 81. Both compounds showed the fragment ion
corresponding to the respective N-allyl-lactam ring of m/z
126 for 9 (N-allyl-lactam five-members ring) and m/z 154
for 10 (N-allyl lactam seven-member ring), formed by neu-
tral loss of the anilines R–Ph–NH2 (Gonçalves et al. 2011).
Differently from the results observed in the EI-MS
study, the ESI-MS results did not show the fragment ions
which correspond to the two loss of water (M−36). Instead,
the compounds showed an interesting pattern with the frag-
ment ions M−46 and M−79. The pattern M−46 was attrib-
uted to a neutral loss of nitrogen dioxide molecule (NO2)
(Gonçalves et al. 2011; Morgan et al. 2014) (Scheme 7)
generating the fragment ions of m/z 377 for compound 9
and m/z 405 for compound 10.
The second pattern observed for the compounds 9–20
was the loss of neutral iminosulfane dioxide molecule
(M−79) which generated the fragment ions m/z 344 for
9 and m/z 372 for 10. This fragmentation pathway occurs
through intramolecular rearrangement displayed in
Scheme 8.
The fragmentation pattern showed for compounds 9 and
10 were observed in the ESI(+)-MS/MS mass spectra of all
synthesized p-(N-alkyl)lactam sulfonamides (9–20).
Biological activity of compounds 1, 3 and 9
A series of N-alkylbenzenesulfonamides were tested for
their effects on potassium ion channels by using electro-
physiological recordings of ionic currents with the patch
clamp technique (Hamill et al. 1981). As shown in Fig. 6,
compounds 1, 3 and 9 are blockers of Kv3.1 channels. In
1 3

C. C. Martins et al.
Scheme 6 Possible pathways
for fragmentation of M−18
species
.
+
O
HN
S
O
O
H
- H2O
(scheme 5)
m/z 387 (for 2)
m/z 415 (for 3)
O
N
N
-
N
O
+
n
.
.
.
+
+
+
.
O
O
O
+
O
CH3
N
HN
S
HN
S
HN
S
O
O
O
O
H
O
O
O
HN
S
_
- H2O
n
O
O
"ortho effect"
+
O
O
O
N
N
N
N
N
NH
O
-
-
O
N
H
N
N
H
O
N
N
m/z 304
O
+
O
n
n
n
2. n = 1, m/z 423
[M - 18]
.
3. n = 3, m/z 451
+
.
O
+
O
H
N
HN
S
O
O
H
O
_
HN
n
O
S
O
O
N
N
-
O
N
N
N
O
+
m/z 320
n
the presence of the drugs, the current rises and then decays
to a new steady state, suggesting an interaction with the
channel in the open state.
Dose–response relationships were constructed for each
of the compounds referred in Fig. 6, the Hill equation was
fitted to the data points and their IC50 and Hill coefficients
are shown in Table 1.
The binding of the molecule to the channel is reversible
upon washout of the drug from the bath solution. Prelimi-
nary studies of the effects of these compounds on Kv1.3
and Kv1.4 shows a much lower affinity than that reported
above for the Kv3.1 channels. Detailed characterization
of the mechanism of action of the compounds on the ion
channels are being carried out.
Conclusions
We showed that the nitro group does not affect the frag-
mentation pattern of compounds (1–8) by ESI(−)-MS/
MS; however, it influences the mechanism of formation
of sulfoxylate with m/z 65. EI-MS spectra for the p-sub-
stituted aminolactams (9–20) showed the loss of two water
molecules, which is associated with the ortho effect pre-
sent in the N-alkyl-ortho-nitroanilines and a McLafferty
rearrangement. ESI(+)-MS/MS spectra of such com-
pounds showed the loss of M−79, which corresponds to
iminosulfane dioxide (SO2NH) and also the loss of nitro
group (M−46). The synthesized molecules are under cur-
rent investigation for their inhibitory activity of potassium
1 3

Mass spectrometry study of N-alkylbenzenesulfonamides with potential antagonist activity to…
Fig. 5 ESI(+)-MS/MS of
CH2
compounds 9 and 10
O
O
Intens.
x10
4
HN
7
5
3
1
423
O
S O
81.03260
N
NO2
O
H
NH
M-46
+
N
M-79
[M+H]
423.12965
O
9
377.13732
344.15781
126.09026
MM = 422 g.mol-1
405.11895
390
430
m/z
70
110
150
190
230
270
310
350
O
CH2
O
Intens.
HN
5
O
S O
x10
N
81.03278
O
NO2
H
NH
2
1
0
N
451
O
10
154.12199
MM = 450 g.mol-1
M-79
M-46
+
[M+H]
451.16132
372.18936
405.16896
m/z
80
160
240
320
400
480
H+
H+
Experimental section
Materials and methods
O
O
HN
O S O
HN
O S O
The reagents used in this work (4-chlorobenzenesulfonic
chloride, N-(3-aminopropyl)-2-pyrrolidone (APP), ben-
zylamine, butylamine, furfurylamine, cyclohexylamine,
4-chlorobenzylamine, 2,4-dichlorobenzylamine, hydro-
chloric acid and solvents: 99 % ethanol, acetonitrile and
dimethylsulfoxide) were all commercially obtained and
used without further purification.
- NO2
+
O
N
NH
O
NH
O
-
General procedure for the synthesis of p‑chloro‑N‑alkylb
enzenesulfonamides (1–8)
N
N
O
9
m/z 377
m/z 423
4-Chloro-3-nitrobenzenesulfonyl chloride (1 equiv.) and
different amines (2 equiv.) were added to a 50 mL round-
bottomed flask and dissolved in acetonitrile (25 mL). The
contents were then stirred for 1–2 h at room temperature.
Afterward, the solvent was removed under vacuum and
the product was washed with a dilute solution (5 %) of
NaHCO3 and filtered.
Scheme 7 Loss of neutral NO2 molecule from compound 9
channels and some molecules have already shown positive
results.
1 3

C. C. Martins et al.
H+
Scheme 8 The proposed
mechanism for the loss of
iminosulfane dioxide molecule
(SO2NH) for compound 9
H+
H+
O
H
N
NH
O S O
O
O
O
O
C
NH
H
S
H2
O
H2
O
S
H2C
C
H
–O
H
–O
N+
–O
N+
O
N+
O
O
NH
O
NH
O
NH
O
N
N
N
m/z 423
- SO2NH
H+
H2
C
O
–O
N+
O
NH
O
N
m/z 344
Fig. 6 Compounds 1 (a), 3 (b) and 9 (c) block ionic currents through
Kv3.1 channels. The currents were elicited by a voltage pulse from
−88 to +32 mV (70 ms). Black lines represent the currents in control
conditions and gray lines are the current responses in the presence of
the particular compound at a concentration of 80 µM
General procedure for the synthesis of p‑(N‑alkyl)lactam
sulfonamides (9–20)
Table 1 Characteristics of sulfonamides 1, 3 and 9 blockade of
Kv3.1 channel
Identification
IC50 (μM)
Hill coefficient
p-Chloro-N-alkylbenzenesulfonamides (1–8) (1 equiv.) and
aminolactam N-(3-aminopropyl)-2-pyrrolidinone (APP)
(1.25 equiv.) or N-(3-aminopropyl)-2-azepanone (APA)
(1.25 equiv.) were added to a 50 mL round-bottomed flask
and dissolved in dimethylsulfoxide (10 mL) for a nucleo-
philic aromatic substitution reaction (SNAr). The contents
Compound 1 (N = 5)
Compound 3 (N = 9)
Compound 9 (N = 5)
16.9 0.6
12.5 0.5
34.3 0.9
1.5 0.06
1.72 0.05
1.61 0.06
N is the number of cells tested in each experiment
1 3

Mass spectrometry study of N-alkylbenzenesulfonamides with potential antagonist activity to…
were then stirred in a reflux system for 1–2 h at 85 °C.
Afterward, the solvent was removed with frozen water and
the product was treated with diluted HCl (5 %) for removal
of amino-lactam excess. The crude product was filtered off
and recrystallized from 1:1 ethyl ether–petroleum ether (bp
60–80 °C). All obtained compounds were structurally con-
electronically compensated up to 80 % when currents
exceeded 2.0 nA. The signal was low-pass filtered at
5.0 kHz and acquired at 20 kHz through an AxoPacth 200B
amplifier (Axon Instruments®, Foster City, CA, USA) con-
nected to an A/D and D/A converter Digidata 1440A (Axon
Instruments®). The data were analyzed with Origin 8.0
software (OriginLab®, Northampton, MA, USA). Chemi-
cals used for electrophysiological studies were purchased
from Sigma (St Louis, MO, USA).
1
firmed by 13C and H NMR analyses (see Supplementary
material).
Methodology of the study by mass spectrometry
4‑Chloro‑3‑nitro‑N‑furfurylbenzenesulfonamide (base
EI-MS spectra of all compounds were obtained by direct
introduction of the samples in the mass spectrometer
GC–MS Shimadzu QP-2010 Plus. The parameters used
for these analyzes were: interface temperature, 240 °C;
ionization chamber temperature, 300 °C; solvent cutting
time, 0.25 min; start time, 0.30 min; end time, 25.0 min.
DI temperature program: initial temperature of 50 °C,
with heating at 20 °C min−1 to 350 °C and hold time of
10 min.
ESI-MS spectra were obtained using a mass spectrom-
eter Agilent ifunnel Q-TOF 6550 LC–MS with source Dual
Agilent Jet Stream ESI (Dual AJS-ESI). The solution of the
samples was directly injected into the ESI source using an
autosampler FIA at flow rate of 1 µL min−1 at 25 °C. The
solutions were eluted in a gradient of 20 % water (0.1 %
formic acid) and 80 % methanol (0.1 % formic acid) for
ESI(+), and gradient 20 % water (0.1 % ammonium ace-
tate) and 80 % methanol (0.1 % ammonium acetate) for
ESI(−). Mass spectra of sulfonamide by ESI-MS and
ESI-MS/MS were acquired using the following operat-
ing conditions: capillary voltage 3 kV, drying gas flow
10 L min−1, drying gas at 250 °C, nebulizer gas at 50 psi.
The system was operated for acquisitions in the mass range
of 50–1500 m/z. For MS/MS experiments, collision energy
varied between 5 and 25 V using N2. Acquisition and data
processing were performed using the Agilent MassHunter
Workstation software (B.06.01 version).
consulted = CSID:2362584) (1)
Beige solid (0.434 g, 68.7 %), mp = 131.8–132.4 °C;
1
MM = 316.17 g mol−1. H NMR (600 MHz, DMSO) δ
8.62 (s, 1H), 8.31 (d, J = 2.1 Hz, 1H), 7.96 (d, J = 2.0 Hz,
1H), 7.95 (s, 1H), 7.40 (dd, J = 1.8, 0.8 Hz, 1H), 6.24 (dd,
J = 3.2, 1.9 Hz, 1H), 6.17 (d, J = 3.2 Hz, 1H), 4.13 (s, 2H).
13C NMR (151 MHz, DMSO) δ 149.87, 147.13, 142.59,
141.09, 132.82, 131.25, 129.01, 123.86, 110.27, 108.51,
39.16. EI-MS m/z found: 316. ESI(−)-MS m/z found:
314.98674, m/z calculated for [C11H9ClN2O5S−H]−:
314.98479.
4‑Chloro‑N‑furfurylbenzenesulfonamide (base con
sulted = CSID:91535) (2)
Brown solid (0.320 g, 59 %), mp = 118.7–119.9 °C;
1
MM = 271.72 g mol−1. H NMR (400 MHz, CDCl3) δ
7.76–7.74 (m, 1H), 7.74–7.72 (m, 1H), 7.45–7.44 (m, 1H),
7.44–7.42 (m, 1H), 7.22 (dd, J = 1.8, 0.7 Hz, 1H), 6.22
(dd, J = 3.2, 1.9 Hz, 1H), 6.09 (dd, J = 3.2, 0.6 Hz, 1H),
4.84 (t, J = 5.5 Hz, 1H), 4.21 (d, J = 6.0 Hz, 2H). 13C
NMR (101 MHz, CDCl3) δ 149.31, 149.30, 142.73, 139.20,
138.69, 138.67, 129.37, 128.64, 110.53, 108.60, 108.59,
40.20. EI-MS m/z found: 271. ESI(−)-MS m/z found:
270.00074, m/z calculated for [C11H10ClNO3S−H]−:
269.99972.
Methodology of the study of biological activity
4‑Chloro‑3‑nitro‑N‑butylbenzenesulfonamide (CAS No.
96‑61‑7) (3)
For the in vitro studies, we used the L-929 cell line sta-
bly expressing Kv 1.3 and 3.1 channels and CHO cell
line, stably expressing Kv1.4. Cells were voltage clamped
at a holding potential of −88 mV and pulsed to +32 mV
(70 ms) to activate the potassium currents, essentially as
described in Rodrigues et al. (2003). The bath solution had
the following composition (in mM): 155 NaCl, 1.0 MgCl2,
2.0 CaCl2, 5.0 HEPES and 4.5 KCl, pH 7.4 (NaOH) and
osmolality of 300–310 mOsm/kg H2O. The pipette solution
was composed of (in mM): 155 KF, 5.0 EGTA, 10 HEPES,
2.0 MgCl2 and 1.0 CaCl2, pH 7.2 (KOH) and osmolal-
ity of 290–300 mOsm/kg H2O. The series resistance was
White yellow solid. (0.450 g, 77 %), mp = 68–69 °C;
1
MM = 292.73 g mol−1. H NMR (400 MHz, DMSO)
δ 8.42 (d, J = 1.8 Hz, 1H), 8.05 (dd, J = 8.5, 2.0 Hz,
1H), 8.02 (d, J = 8.3 Hz, 1H), 7.95 (t, J = 5.8 Hz, 1H),
2.80 (dd, J = 12.8, 6.9 Hz, 2H), 1.40–1.32 (m, 2H),
1.24 (sext, J = 14.0, 7.0 Hz, 2H), 0.81 (t, J = 7.3 Hz,
3H). 13C NMR (101 MHz, DMSO) δ 147.45, 140.88,
133.09, 131.26, 129.12, 123.86, 42.21, 31.05, 19.12,
13.40. EI-MS m/z found: 392. ESI(−)-MS m/z found:
291.02251, m/z calculated for [C10H13ClN2O4S−H]−:
291.02118.
1 3

C. C. Martins et al.
4‑Chloro‑N‑butylbenzenesulfonamide (CAS No. 6419‑73‑4)
4‑Chloro‑3‑nitro‑N‑2,4‑dichlorobenzylbenzenesulfonamide
(4)
(8)
White solid. (0.164 g, 33.1 %); mp = 46–46.5 °C;
Beige solid (0.483 g, 61 %), mp = 135–136.5 °C;
1
1
MM = 247.74 g mol−1. H NMR (400 MHz, CDCl3) δ
MM = 395.65 g mol−1. H NMR (400 MHz, CDCl3) δ
7.84–7.76 (m, 2H), 7.53–7.44 (m, 2H), 4.81 (t, J = 5.9 Hz,
1H), 2.93 (dd, J = 13.3, 6.9 Hz, 2H), 1.47–1.40 (m, 2H),
1.32–1.23 (m, 2H), 0.84 (t, J = 7.3 Hz, 3H). 13C NMR
(101 MHz, CDCl3) δ 139.16, 138.65, 129.49, 128.64,
43.07, 31.67, 19.78, 13.62. EI-MS m/z found: 247.
ESI(−)-MS m/z found: 246.03720, m/z calculated for
[C10H14ClNO2S−H]−: 246.0361.
8.18 (d, J = 2.2 Hz, 1H), 7.86–7.83 (m, 1H), 7.62 (d,
J = 8.4 Hz, 1H), 7.28 (d, J = 2.0 Hz, 1H), 7.24 (s, 1H),
7.19 (dd, J = 8.2, 2.0 Hz, 1H), 5.21 (t, J = 6.2 Hz, 1H),
4.33 (d, J = 6.4 Hz, 2H). 13C NMR (101 MHz, CDCl3) δ
140.65, 135.44, 134.33, 132.90, 131.75, 131.71, 131.61,
130.88, 129.70, 127.65, 124.45, 45.14. EI-MS m/z found:
395. ESI(−)-MS m/z found: 394.92664, m/z calculated for
[C13H9Cl3N2O4S−H]−: 392.92758.
4‑Chloro‑3‑nitro‑N‑benzylbenzenesulfonamide (CAS No.
82835‑65‑2) (5)
4‑[N‑(3′‑Aminopropyl)‑2‑pyrrolidone]‑3‑nitro‑N‑furfurylb
enzenesulfonamide (9)
Yellow solid (0.465 g, 71.2 %), mp = 93–95 °C;
1
MM = 326.75 g mol−1. H NMR (600 MHz, DMSO) δ
Yellow solid (0.463 g, 54.8 %), mp = 115–117.8 °C;
1
8.60 (t, J = 6.3 Hz, 1H), 8.30 (d, J = 2.1 Hz, 1H), 7.98 (dd,
J = 8.4, 2.1 Hz, 1H), 7.92 (d, J = 8.4 Hz, 1H), 7.25–7.15
(m, 5H), 4.10 (d, J = 6.2 Hz, 2H). 13C NMR (151 MHz,
DMSO) δ 147.13, 141.17, 136.85, 132.94, 131.31, 129.09,
128.19, 127.80, 127.23, 123.98, 46.22. EI-MS m/z found:
325. ESI(−)-MS m/z found: 325.00784, m/z calculated for
[C13H11ClN2O4S−H]−; 325.00553.
MM = 422.45 g mol−1. H NMR (600 MHz, DMSO) δ
8.64 (t, J = 6.0 Hz, 1H), 8.31 (d, J = 2.3 Hz, 1H), 8.19
(t, J = 6.4 Hz, 1H), 7.71 (dd, J = 9.2, 2.3 Hz, 1H), 7.29
(d, J = 8.5 Hz, 2H), 7.22 (d, J = 8.4 Hz, 2H), 7.17 (d,
J = 16.1 Hz, 1H), 4.01 (d, J = 6.3 Hz, 2H), 3.44–3.39
(m, 2H), 3.38–3.36 (m, 2H), 3.28 (t, J = 6.7 Hz, 2H),
2.24 (t, J = 8.1 Hz, 2H), 1.98–1.90 (m, 2H), 1.79 (qu,
J = 6.7 Hz, 2H). 13C NMR (151 MHz, DMSO) δ 174.71,
150.74, 147.04, 142.90, 133.65, 130.16, 126.51, 115.77,
110.72, 108.55, 46.83, 40.53, 39.77, 39.67, 30.88, 26.33,
18.00. EI-MS m/z found: 423. ESI(+)-MS m/z found:
23.13222, m/z calculated for [C18H22N4O6S+H]+:
423.13273.
4‑Chloro‑3‑nitro‑N‑cyclohexylbenzenesulfonamide (base
consulted = CSID:5876919) (6)
Yellow solid (0.473 g, 74.4 %), mp = 39–40 °C;
1
MM = 318.04 g mol−1. H NMR (400 MHz, DMSO)
δ 8.45 (d, J = 2.1 Hz, 1H), 8.09–8.06 (m, 1H), 8.02 (t,
J = 7.4 Hz, 2H), 3.08–3.00 (m, 1H), 1.59 (d, J = 9.5 Hz,
4H), 1.19–1.02 (m, 5H). 13C NMR (101 MHz, DMSO)
4‑[N‑(3′‑Aminopropyl)‑2‑azepanone]‑3‑nitro‑N‑furfurylbe
nzenesulfonamide (10)
δ
147.36, 142.57, 133.10, 131.01, 128.97, 123.66,
52.25, 33.18, 24.74, 24.23. EI-MS m/z found: 318.
ESI(−)-MS m/z found: 317.03874, m/z calculated for
[C12H15ClN2O4S−H]−: 317.03683.
Yellow solid (0.381 g, 42.3 %), mp = 145–148 °C;
1
MM = 450.50 g mol−1. H NMR (600 MHz, DMSO-d6)
δ 8.69 (t, J = 6.0 Hz, 1H), 8.35 (d, J = 2.3 Hz, 1H), 8.13
(t, J = 6.1 Hz, 1H), 7.73 (dd, J = 9.1, 2.3 Hz, 1H), 7.43
(dd, J = 1.8, 0.8 Hz, 1H), 7.17 (d, J = 9.3 Hz, 1H), 6.26
(dd, J = 3.2, 1.8 Hz, 1H), 6.16 (dd, J = 3.2, 0.7 Hz, 1H),
4.01 (d, J = 6.1 Hz, 2H), 3.40–3.35 (m, 6H), 2.46–2.41
(m, 2H), 1.74 (qu, J = 6.8 Hz, 2H), 1.66 (qu, J = 5.8 Hz,
2H), 1.55 (dq, J = 11.9, 7.0, 6.4 Hz, 4H). 13C NMR
(151 MHz, DMSO) δ 174.96, 145.05, 135.13, 133.97,
131.58, 130.75, 129.29, 125.03, 123.13, 122.52, 114.03,
48.54, 44.75, 40.38, 39.86, 36.46, 29.22, 28.32, 26.84,
23.02. EI-MS m/z found: 451. ESI(+)-MS m/z found:
451.16137, m/z calculated for [C20H26N4O6S+H]+:
451.16458.
4‑Chloro‑3‑nitro‑N‑4‑chlorobenzylbenzenesulfonamide (7)
Yellow solid (0.500 g, 69.2 %), mp = 107–109 °C;
1
MM = 361.20 g mol−1. H NMR (600 MHz, DMSO)
δ 8.71–8.65 (m, 1H), 8.29 (s, 1H), 8.00–7.95 (m, 1H),
7.92 (d, J = 8.4 Hz, 1H), 7.30–7.24 (m, 2H), 7.21 (d,
J = 8.5 Hz, 2H), 4.09 (d, J = 6.0 Hz, 2H). 13C NMR
(151 MHz, DMSO) δ 147.57, 141.53, 136.43, 133.41,
132.39, 132.03, 131.77, 131.19, 130.08, 129.62, 128.59,
124.43, 123.01, 45.88. EI-MS m/z found: 360. ESI(−)-MS
m/z found: 358.96628, m/z calculated for [C13H11Cl2N2O4
S−H]−: 358.96656.
1 3

Mass spectrometry study of N-alkylbenzenesulfonamides with potential antagonist activity to…
4‑[N‑(3′‑Aminopropyl)‑2‑pyrrolidone]‑3‑nitro‑N‑butylbenz
enesulfonamide (11)
4‑[N‑(3′‑Aminopropyl)‑2‑azepanone]‑3‑nitro‑N‑benzylben
zenesulfonamide (14)
Yellow solid (0.414 g, 52 %), mp = 111–113.7 °C;
Yellow solid (0.389 g, 42.2 %), mp = 157.4–160 °C;
1
1
MM = 398.47 g mol−1. H NMR (600 MHz, DMSO)
MM = 460.54 g mol−1. H NMR (400 MHz, DMSO) δ
δ 8.63 (t, J = 5.9 Hz, 1H), 8.42 (d, J = 2.2 Hz, 1H),
7.78 (dd, J = 9.1, 1.9 Hz, 1H), 7.52 (t, J = 5.8 Hz, 1H),
7.24 (d, J = 9.2 Hz, 1H), 3.40 (q, J = 6.6 Hz, 2H), 3.35
(d, J = 6.9 Hz, 2H), 3.27 (t, J = 6.6 Hz, 2H), 2.71 (dd,
J = 13.1, 6.8 Hz, 2H), 2.22 (t, J = 8.1 Hz, 2H), 1.95–1.89
(m, 2H), 1.79 (qu, J = 6.7 Hz, 2H), 1.37–1.32 (m, 2H),
1.26–1.20 (m, 2H), 0.79 (t, J = 7.3 Hz, 3H). 13C NMR
(151 MHz, DMSO) δ 174.22, 146.63, 133.21, 129.79,
126.01, 125.90, 115.54, 46.34, 42.14, 40.10, 39.30, 30.99,
30.40, 25.86, 19.22, 17.53, 13.43. ESI(+)-MS m/z found:
399.16556, m/z calculated for [C17H26N4O5S+H]+:
399.16967.
8.68 (t, J = 6.0 Hz, 1H), 8.33 (d, J = 2.2 Hz, 1H), 8.12
(t, J = 6.4 Hz, 1H), 7.73 (dd, J = 9.2, 2.1 Hz, 1H), 7.25–
7.15 (m, 6H), 3.99 (d, J = 6.3 Hz, 2H), 3.38 (dd, J = 12.1,
6.3 Hz, 6H), 2.49–2.40 (m, 2H), 1.74 (qu, J = 6.6 Hz, 2H),
1.66 (d, J = 5.0 Hz, 2H), 1.62–1.49 (m, 4H). 13C NMR
(101 MHz, DMSO) δ 175.07, 146.57, 137.29, 133.14,
129.68, 128.11, 127.67, 126.96, 126.14, 115.34, 48.55,
46.15, 44.65, 36.49, 29.27, 28.36, 26.75, 23.04. EI-MS m/z
found: 461. ESI(+)-MS m/z found: 461.18232, m/z calcu-
lated for [C22H28N4O5S+H]+: 461.18532).
4‑[N‑(3′‑Aminopropyl)‑2‑pyrrolidone]‑3‑nitro‑N‑cyclohexy
lbenzenesulfonamide (15)
4‑[N‑(3′‑Aminopropyl)‑2‑azepanone]‑3‑nitro‑N‑butylbenze
nesulfonamide (12)
Yellow solid (0.356 g, 42 %), mp = 148–151.8 °C;
1
MM = 424.51 g mol−1. H NMR (600 MHz, DMSO) δ
Yellow solid (0.272 g, 31.9 %), mp = 124.4–126 °C;
8.62 (t, J = 6.0 Hz, 1H), 8.45 (d, J = 2.3 Hz, 1H), 7.82
(dd, J = 9.2, 2.2 Hz, 1H), 7.62 (d, J = 7.2 Hz, 1H), 7.25
(d, J = 9.3 Hz, 1H), 3.41 (dd, J = 13.2, 6.6 Hz, 2H),
3.37–3.34 (m, 2H), 3.28 (t, J = 6.7 Hz, 2H), 2.92 (d,
J = 6.7 Hz, 1H), 2.22 (t, J = 8.1 Hz, 2H), 1.97–1.88 (m,
2H), 1.80 (qu, J = 6.8 Hz, 2H), 1.60 (dd, J = 10.2, 5.2 Hz,
4H), 1.14 (dd, J = 13.7, 8.9 Hz, 4H). 13C NMR (151 MHz,
DMSO) δ 174.26, 146.58, 136.38, 133.11, 131.67, 129.66,
129.52, 128.04, 126.16, 126.12, 115.37, 46.37, 45.41,
40.05, 39.30, 30.42, 25.93, 17.54. EI-MS m/z found: 425.
ESI(+)-MS m/z found: 425.18190, m/z calculated for
[C19H28N4O5S+H]+: 425.18532.
1
MM = 426.53 g mol−1. H NMR (400 MHz, DMSO) δ
8.69 (t, J = 6.0 Hz, 1H), 8.42 (d, J = 2.2 Hz, 1H), 7.78
(dd, J = 9.1, 2.1 Hz, 1H), 7.51 (t, J = 5.8 Hz, 1H), 7.24
(d, J = 9.3 Hz, 1H), 3.38 (dt, J = 9.4, 6.4 Hz, 6H), 2.71
(dd, J = 13.0, 6.8 Hz, 2H), 2.45–2.40 (m, 2H), 1.75 (qu,
J = 6.6 Hz, 2H), 1.70–1.62 (m, 2H), 1.55 (d, J = 4.2 Hz,
4H), 1.39–1.31 (m, 2H), 1.23 (dd, J = 15.0, 7.2 Hz, 2H),
0.80 (t, J = 7.3 Hz, 3H). 13C NMR (101 MHz, DMSO) δ
175.03, 146.63, 133.20, 129.79, 125.92, 115.50, 48.53,
44.67, 42.13, 36.48, 30.98, 29.26, 28.34, 26.74, 23.03,
19.26, 13.45. EI-MS m/z found: 427. ESI(+)-MS m/z
found: 427.19950, m/z calculated for [C19H30N4O5S+H]+:
427.20097.
4‑[N‑(3′‑Aminopropyl)‑2‑azepanone]‑3‑nitro‑N‑cyclohexyl
benzenesulfonamide (16)
4‑[N‑(3′‑Aminopropyl)‑2‑pyrrolidone]‑3‑nitro‑N‑benzylbe
nzenesulfonamide (13)
Yellow solid (0.430 g, 47.5 %), mp = 156.3–159.1 °C;
1
MM = 452.56 g mol−1. H NMR (600 MHz, DMSO) δ
Yellow solid (0.441 g, 51 %), mp = 151–153.9 °C;
8.62 (t, J = 6.0 Hz, 1H), 8.45 (dd, J = 5.6, 3.7 Hz, 1H),
7.85–7.77 (m, 1H), 7.62 (d, J = 7.2 Hz, 1H), 7.25 (d,
J = 9.3 Hz, 1H), 3.39 (d, J = 6.6 Hz, 5H), 3.36–3.33 (m,
1H), 3.27 (d, J = 6.7 Hz, 1H), 2.91 (s, 1H), 2.23 (s, 1H),
1.94 (s, 1H), 1.83–1.76 (m, 1H), 1.59 (dd, J = 10.1, 5.4 Hz,
1H), 1.13 (d, J = 10.9 Hz, 1H). 13C NMR (101 MHz,
DMSO) δ 175.06, 147.40, 142.67, 133.16, 131.12, 129.03,
123.73, 54.15, 52.35, 48.56, 45.39, 44.67, 36.49, 33.24,
29.26, 28.36, 26.73, 24.80, 24.31, 23.04. EI-MS m/z found:
453. ESI(+)-MS m/z found: 453.21379, m/z calculated for
[C21H32N4O5S+H]+: 453.21662.
1
MM = 432.49 g mol−1. H NMR (600 MHz, DMSO) δ
8.62 (t, J = 6.0 Hz, 1H), 8.32 (d, J = 2.4 Hz, 1H), 8.13
(t, J = 6.4 Hz, 1H), 7.73 (dd, J = 9.1, 2.2 Hz, 1H), 7.25–
7.15 (m, 6H), 3.99 (d, J = 6.5 Hz, 2H), 3.40–3.34 (m, 4H),
3.27 (dt, J = 6.7, 3.3 Hz, 2H), 2.23 (t, J = 8.0 Hz, 2H),
1.96–1.90 (m, 2H), 1.78 (qu, J = 6.8 Hz, 2H). 13C NMR
(151 MHz, DMSO) δ 174.27, 146.57, 137.28, 133.15,
129.69, 128.11, 127.67, 126.96, 126.12, 115.37, 46.37,
40.05, 39.30, 30.42, 25.87, 17.55. EI-MS m/z found: 433.
ESI(+)-MS m/z found: 433.15016, m/z calculated for
[C20H24N4O5S+H]+: 433.15402.
1 3

C. C. Martins et al.
4‑[N‑(3′‑Aminopropyl)‑2‑pyrrolidone]‑3‑nitro‑N‑4‑chlorob
enzylbenzenesulfonamide (17)
4‑[N‑(3′‑Aminopropyl)‑2‑azepanone]‑3‑nitro‑N‑2,4‑dichlo
robenzylbenzenesulfonamide (20)
Yellow solid (0.361 g, 38.7 %), mp = 167.7–170.5 °C;
MM = 466.93. 1H NMR (600 MHz, DMSO) δ 8.64
(t, J = 6.0 Hz, 1H), 8.31 (d, J = 2.3 Hz, 1H), 8.19 (t,
J = 6.4 Hz, 1H), 7.71 (dd, J = 9.2, 2.3 Hz, 1H), 7.29 (d,
J = 8.5 Hz, 2H), 7.22 (d, J = 8.4 Hz, 2H), 7.16 (s, 1H),
4.01 (d, J = 6.3 Hz, 2H), 3.43–3.39 (m, 2H), 3.38–3.36 (m,
2H), 3.28 (t, J = 6.7 Hz, 2H), 2.24 (t, J = 8.1 Hz, 2H),
1.99–1.90 (m, 2H), 1.79 (qu, J = 6.7 Hz, 2H). 13C NMR
(151 MHz, DMSO) δ 174.26, 146.58, 136.38, 133.11,
131.67, 129.66, 129.52, 128.04, 126.16, 126.12, 115.37,
46.37, 45.41, 40.05, 39.30, 30.42, 25.93, 17.54. EI-MS m/z
found: 467. ESI(+)-MS m/z found: 467.11199, m/z calcu-
lated for [C20H23N4ClO5S+H]+: 467.11504.
Yellow solid (0.497 g, 46.9 %), mp = 154–160 °C;
1
MM = 529.44 g mol−1. H NMR (400 MHz, CDCl3) δ
8.64 (t, J = 5.2 Hz, 1H), 8.56 (d, J = 2.3 Hz, 1H), 7.69
(dd, J = 9.1, 2.3 Hz, 1H), 7.29 (d, J = 8.3 Hz, 1H), 7.16
(dd, J = 8.2, 2.1 Hz, 1H), 6.83 (d, J = 9.2 Hz, 1H), 5.27
(t, J = 6.5 Hz, 1H), 4.26 (d, J = 6.3 Hz, 2H), 3.53 (t,
J = 6.4 Hz, 2H), 3.41–3.36 (m, 4H), 2.58–2.53 (m, 2H),
1.90 (dd, J = 13.1, 6.6 Hz, 2H), 1.77–1.64 (m, 6H). 13C
NMR (101 MHz, CDCl3) δ 177.00, 147.14, 133.50, 132.68,
131.63, 131.58, 130.95, 129.43, 127.58, 127.42, 126.17,
124.48, 114.27, 50.04, 45.84, 44.81, 40.64, 37.16, 30.05,
28.76, 27.44, 23.49. EI-MS m/z found: 529. ESI(+)-MS
m/z found: 529.10526, m/z calculated for [C22H26Cl2N4O5
S+H]+: 529.10737.
4‑[N‑(3′‑aminopropyl)‑2‑azepanone]‑3‑nitro‑N‑4‑chlorobe
nzylbenzenesulfonamide (18)
Acknowledgments The authors would like to thank the Brazil-
ian Research Foundations FAPESP (research fund 2013/24487-6),
CNPq and CAPES for their financial assistance. We are also grateful
to Post-graduation Program POSMAT at São Paulo State University
(UNESP) for the post-graduation fellowship.
Yellow solid (0.368 g, 37.2 %), mp = 165.6–171.7 °C;
1
MM = 494.99 g mol−1. H NMR (400 MHz, DMSO) δ
8.71 (t, J = 6.0 Hz, 1H), 8.33 (d, J = 2.2 Hz, 1H), 8.19
(t, J = 6.4 Hz, 1H), 7.72 (dd, J = 9.2, 2.2 Hz, 1H), 7.30
(d, J = 8.5 Hz, 2H), 7.24 (d, J = 8.4 Hz, 2H), 7.17 (d,
J = 9.3 Hz, 1H), 4.02 (d, J = 6.4 Hz, 2H), 3.40 (qua,
J = 6.1 Hz, 6H), 2.49–2.42 (m, 2H), 1.76 (qu, J = 6.6 Hz,
2H), 1.68 (d, J = 5.0 Hz, 2H), 1.58 (dd, J = 9.1, 4.4 Hz,
4H). 13C NMR (101 MHz, DMSO) δ 175.04, 146.57,
136.39, 133.09, 131.64, 129.64, 129.60, 129.51, 128.14,
128.03, 126.13, 126.06, 115.33, 48.55, 45.38, 44.67,
36.48, 29.26, 28.35, 26.73, 23.03. EI-MS m/z found: 487.
ESI(+)-MS m/z found: 495.14268, m/z calculated for
[C22H27ClN4O5S+H]+: 495.14634.
Compliance with ethical standards
Conflict of interest The authors declare that they have no competing
financial interests.
References
Bromidge SM, Charke SE, King FD, Lovell PJ, Newman H, Riley G,
Routledge C, Serafinowska HT, Smith DR, Thomas DR (2002)
Bicyclic piperazinylbenzenesulphonamides are potent and
selective 5-HT6 receptor antagonists. Bioorg Med Chem Lett
12(10):1357–1360. doi:10.1016/S0960-894X(02)00172-5
Danikiewicz W (1998) Electron ionization-induced fragmentation
of N‑alkyl-o-nitroanilines: observation of new types of ortho
effects. Eur J Mass Spectrom 4:167
Dannhardt G, Kiefer W (2001) Cyclooxygenase inhibitors—current
status and future prospects. Eur J Med Chem 36(2):109–126.
doi:10.1016/S0223-5234(01)01197-7
Deng X, Mani NS (2006) A facile, environmentally benign sulfona-
mide synthesis in water. Green Chem 8:835–838. doi:10.1039/
b606127c
Fiedorow P, Franski R, Gierczyk B, Schroeder G (2005) Fragmen-
tation and skeletal rearrangements of products of the reaction
between fluorobenzenes and bicyclic N-bases studied by electron
ionization mass spectrometry. Int J Mass Spectrom 240:7–15.
doi:10.1016/j.ijms.2004.10.001
Ghosh AK, Leshchenko-Yashchuk S, Anderson DD, Baldridge A,
Noetzel M, Miller HB, Tie Y, Wang Y, Koh Y, Weber IT, Mit-
suya H (2009) Design of HIV-1 protease inhibitors with pyr-
rolidinones and oxazolidinones as novel P1′-ligands to enhance
backbone-binding interactions with protease: synthesis, biologi-
cal evaluation, and protein-ligand X-ray studies. J Med Chem
52(13):3902–3914. doi:10.1021/jm900303m
4‑[N‑(3′‑Aminopropyl)‑2‑pyrrolidone]‑3‑nitro‑N‑2,4‑dichl
orobenzylbenzenesulfonamide (19)
Yellow solid (0.270 g, 27 %); mp = 142.2–145 °C;
1
MM = 501.38 g mol−1. H NMR (400 MHz, CDCl3) δ
8.55 (d, J = 2.2 Hz, 1H), 8.51 (t, J = 4.8 Hz, 1H), 7.68
(dd, J = 9.1, 2.2 Hz, 1H), 7.27 (s, 1H), 7.24 (s, 1H), 7.15
(dd, J = 8.2, 2.1 Hz, 1H), 6.81 (d, J = 9.1 Hz, 1H), 5.10
(t, J = 6.5 Hz, 1H), 4.25 (d, J = 6.4 Hz, 2H), 3.46–3.35
(m, 6H), 2.41 (t, J = 8.1 Hz, 2H), 2.05 (dd, J = 15.1,
7.6 Hz, 2H), 1.93 (dd, J = 13.3, 6.7 Hz, 2H). 13C NMR
(101 MHz, CDCl3) δ 176.09, 147.08, 134.55, 134.20,
133.57, 132.65, 131.54, 130.78, 129.40, 127.55, 127.42,
126.43, 114.31, 47.63, 44.78, 40.88, 40.40, 30.97, 26.74,
18.10. EI-MS m/z found: 501. ESI(+)-MS m/z found:
501.07316, m/z calculated for [C20H22Cl2N4O5S+H]+:
501.07607.
Gonçalves RS, Abdelnur PV, Santos VG, Simas RC, Eberlin MN,
Magalhães A, Gonzalez ERP (2011) Synthesis of potentially
1 3

Mass spectrometry study of N-alkylbenzenesulfonamides with potential antagonist activity to…
bioactive PABA-related N-(aminoalkyl)lactamic amino acids and
esters via selective SNAr reactions. Amino Acids 40(1):197–204.
doi:10.1007/s00726-010-0634-z
Morgan KM, Ashline DJ, Morgan JP, Greenberg A (2014) Electro-
spray ionization (ESI) fragmentations and Dimethyldioxirane
reactivities of three diverse lactams having full, half, and zero
resonance energies. J Org Chem 79(2):517–528
Goodman A (1996) As bases farmacológicas da terapêutica. McGraw
Hill, Cidade do México, p 1436
Gouauax E, Mackinnon R (2005) Principles of selective ion transport
in channels and pumps. Science, Washington 310(5753):1461–
1465. doi:10.1126/science.1113666
Gross MF, Beaudoin S, McNaughton-Smith G, Amato GS, Castle NA,
Huang C, Zou A, Yu W (2007) Aryl sulphonamido indane inhibi-
tors of the Kv1.5 ion channel. Med Chem Lett 17(10):2849–
2853. doi:10.1016/j.bmcl.2007.02.052
Olsson RI, Jacobson I, Iliefski T, Boström J, Davidsson Ö, Fjell-
ström O, Björe A, Olsson C, Sundell J, Gran U, Gyll J, Malm-
berg J, Hidestål O, Emtenäs H, Svensson T, Yuan Z, Strandlund
G, Åstrand A, Lindhardt E, Linhardt G, Forsström E, Högberg
Å, Persson F, Andersson B, Rönnborg A, Löfberg B (2014)
Lactam sulfonamides as potent inhibitors of the Kv1.5 potas-
sium ion channel. Bioorg Med Chem Lett 24(5):1269–1273.
doi:10.1016/j.bmcl.2014.01.067
Hamill OP, Marty A, Neher E, Sakmann B, Sigworth FJ (1981)
Improved patch-clamp techniques for high resolution current
recording from cells and cell-free membrane patch. Pfluegers
Arch 391(2):85–100. doi:10.1007/BF00656997
Rodrigues ARA, Arantes EC, Monje F, Stuhmer W, Varanda WA
(2003) Tityustoxin-K(alpha) blockade of the voltage-gated
potassium channel Kv1.3. Br J Pharmacol 139(6):1180–1186.
doi:10.1038/sj.bjp.0705343
Hibbs JA, Jariwala FB, Weisbecker CS, Attygalle ABJ (2013) Gas-
phase fragmentations of anions derived from N-phenyl ben-
zenesulfonamides. Am Soc Mass Spectrom 24:1280–1287.
doi:10.1007/s13361-013-0671-4
Scozzafava A, Mastrolorenzo A, Supuran CT (2002) Arylsulfo-
nyl-N,N-diethyldithiocarbamates: a novel class of antitumor
agents. Bioorg Med Chem Lett 10:1887–1891. doi:10.1016/
S0960-894X(00)00375-9
Hu N, Liu P, Jiang K, Zhou Y, Pan Y (2008) Mechanism study of
SO2 elimination from sulfonamides by negative electrospray
ionization mass spectrometry. Rapid Commun Mass Spectrom
22:2715–2722. doi:10.1002/rcm.3672
Smith MB, March J (2001) March’s advanced organic chemistry.
Wiley, New York
Sun M, Dai W, Liu DQ (2007) Fragmentation of aromatic sulfona-
mides in electrospray ionization mass spectrometry: elimina-
tion of SO2
doi:10.1002/jms.1335
Hu N, Tu Y, Jiang K, Pan Y (2010) Intramolecular charge transfer
in the gas phase: fragmentation of protonated sulfonamides in
mass spectrometry. J Org Chem 74:4244–4250. doi:10.1007/
s13361-014-0935-7
Irikura KK, Todua NG (2014) Facile Smiles-type rearrangement in
radical cations of N-acylarylsulfonamides and analogs. Rapid
Commun Mass Spectrom 28:829–834. doi:10.1002/rcm.6850
Joshi S, Khosla N (2003) QSAR study on antibacterial activity of sul-
phonamides and derived Mannich bases. Bioorg Med Chem Lett
13:3747–3751. doi:10.1016/j.bmcl.2003.08.017
via rearrangement. J Mass Spectrom 43:383–393.
Supuran CT, Scozzafava A, Jurca BC, Ilies MA (1998) Carbonic
anhydrase inhibitors—part 49: synthesis of substituted ureido
and thioureido derivatives of aromatic/heterocyclic sulfonamides
with increased affinities for isozyme I. Eur J Med Chem 33:83–
93. doi:10.1016/S0223-5234(98)80033-0
Wulf H, Castle NA, Pardo LA (2009) Voltage-gated potassium
channels as therapeutic targets. Nat Rev Drug Discov Lond
8(12):982–999. doi:10.1038/nrd2983
Li JJ, Anderson QD, Burton EG, Cogburn JN, Collins JT, Garland DJ,
Gregory SA, Huang HC, Isakson PC, Kobaldt CM, Longush EN,
Norton MB, Perkns WE, Reinhard EJ, Serbert K, Veenhuizen
AW, Zang Y, Rertz DB (1995) 1,2-Diarylcyclopentenes as selec-
tive cyclooxygenase-2 inhibitors and orally active antiinflamma-
tory agents. J Med Chem 38(22):4570–4578
Lloyd J, Atwal KS, Finlay HJ, Nyman M, Huynh T, Bhandaru
R, Kover A, Schmidt J, Vaccaro W, Conder ML, Jenkins-
West T, Levesque P (2007) Benzopyran sulphonamides as
Kv1.5 potassium channel blockers. Bioorg Med Chem Lett
17(12):3271–3275. doi:10.1016/j.bmcl.2007.04.020
Yamaoka H, Kusagi I, Isa K, Maekawa Y, Nibbering NMM (2004)
On the double benzylic hydrogen migration in the molecular ion
of N-(5-phenylvaleryl)-1-azacyclopentane-2-thione. Int J Mass
Spectrom 234:171
Ye D, Wang J, Yu K, Zhou Y, Jiang H, Chen K, Liu H (2009) Current
strategies for the discovery of K+ channel modulators. Curr Top
Med Chem 9:348–361. doi:10.2174/156802609788317865
Zhao G, Bolton SA, Kwon C, Hartl KS, Seiler SM, Slusarchyk WA,
Sutton JC, Bisacchi GS (2004) Synthesis of potent and selective
2-azepanone inhibitors of human tryptase. Bioorg Med Chem
Lett 14:309–312. doi:10.1016/j.bmcl.2003.11.016
Lü Q, Frank W (2008) Impact of novel screening technologies on ion
channel drug discovery. Comb Chem High Throughput Screen
11(3):185–194. doi:10.2174/138620708783877735
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